This project is concerned with signals responsible for changes in expression and transport function of ATP-driven, xenobiotic efflux pumps (Mrp2, Mrp4, BCRP and p-glycoprotein) in renal proximal tubule. We use comparative models (single, intact proximal tubules from teleost fish, mammalian renal slices, and renal cells in culture) in combination with confocal microscopy to identify the physiologically relevant extracellular signals (hormones, metabolites, xenobiotics), and the intracellular signaling pathways that modulate xenobiotic excretion in proximal tubule. Our experiments show common signaling pathways for short-term (minutes) and long-term (hours to days) regulation of Mrp2 and p-glycoprotein. In the short-term, endothelin-1 (ET-1), acting through a basolateral, B-type ET receptor, a G-protein, nitric oxide synthase (NOS), NO, protein kinase G (PKG), cGMP and PKC, rapidly (minutes) reduces transport mediated by the luminal ATP-driven xenobiotic export pumps, p-glycoprotein and Mrp2;although pump activity decreases, protein expression is unchanged. A parallel signaling system is activated by parathyroid hormone (PTH) and other calciotropic hormones acting through PKC and cGMP. The effects of ET-1 and PTH on Mrp2-mediated transport are additive. Importantly, three structurally unrelated classes of nephrotoxicants (radiocontrast agents, aminoglycoside antibiotics and heavy metal salts) release ET-1 from the tubules and fire the ETB receptor-NOS-cGMP-PKG-PKC signaling pathway. This appears to be an early, common event in nephrotoxicant action that links ET-1 signaling and tubular toxicity. Over the longer-term, (hours to days) sub-toxic concentrations of ET-1 and nephrotoxicants upregulate transport mediated by Mrp2 and increase the Mrp2 protein content of the epithelial cell luminal membranes. Upregulation of transport and transporter expression is abolished when signaling is blocked by inhibitors of NO synthase and PKC. In vitro exposure of tubules to low concentrations of arsenite and in vivo exposure of fish to environmentally relevant water arsenite concentrations also increased renal Mrp2 protein expression and transport activity. Surprisingly, we could find no increase in Mrp2 mRNA with arsenite exposure, suggesting modulation by post-transcriptional mechanisms. Upregulation of Mrp2 by ET-1, gentamicin or arsenite was accompanied by increased tolerance to acute exposure to nephrotoxicants and arsenite, suggesting "preconditioning" causes selective transcriptional activation of protective pathways, likely including xenobiotic efflux. Finally, new experiments have identified two additional modes of transporter regulation in renal proximal tubules. First, ligands of the aryl hydrocarbon receptor (AhR) upregulate P-glycoprotein, Mrp2 and BCRP expression over a period of hours. This appears to be a protective mechanism through which polycyclic aromatics, including dioxin, at very low levels, can induce efflux transport of their polar metabolites. However, such induction has the potential to alter the pharmacokinetics of other toxicants and of therapeutic drugs. We are examining the possibility that a similar regulatory mechanism is active in other barrier and excretory tissues. Second, activation of the glucocorticoid receptor (GR) rapidly increases Mrp2 transport activity by a non-genomic mechanism involving tyrosine kinase-based signaling. This signaling from GR to the transporter could underlie drug-drug interactions at the level of renal efflux, reducing plasma levels of toxicants (a plus) and of therapeutics (a problem).